Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
1994-09-02
2001-10-02
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C435S023000, C435S184000
Reexamination Certificate
active
06297023
ABSTRACT:
BACKGROUND OF THE INVENTION
Plasminogen activator inhibitor-1 is a natural inhibitor of tissue plasminogen activator. Deficiencies in plasminogen activator inhibitor-1 (PAI-1) results in delayed, or prolonged, bleeding. A high level of PAI-1 is indicative of heart disease. A reliable assay for the detection of low or high levels of PAI-1 activity is desirable.
PAI-1 has been expressed as arbitrary units (AU), wherein 1 AU of PAI-1 is the amount that inhibits 1 unit of tissue plasminogen activator (t-PA) under the conditions given. For example, in the assay as sold by Kabi the AU is measured during a 10 minute incubation of samples with standard t-PA at 37° C.
A current method (Coatest®, Kabi Diagnostics) for assaying PAI-1 activity in plasma or serum involves adding 40 U/ml t-PA, incubating the mixture for 10 minutes at room temperature and measuring the remaining t-PA activity in the sample. The remaining t-PA activity is converted to PAI-1 activity (AU/ml) using a standard curve which is generated by adding 40 U/ml t-PA in PAI-1 depleted plasma (0 AU/ml PAI-1) and no t-PA added (40 AU/ml PAI-1). Chmielewska et al.,
Clinical Chemistry
, 32(3):482-485 (1986), incorporated herein by reference.
This prior art method has several drawbacks. The sensitivity or experimental error of the assay near the low and high ends of the range, in this case, 0 and 40+AU/ml (dictated by the amount of t-PA added to the sample), can be poor. For example, samples containing higher activity than 40 AU/ml PAI-1 are not detected, requiring sample dilution or increased amounts of t-PA to be added to the assay with the development of new standard curves.
PAI-1, t-PA and the complex of PAI-1·t-PA are under equilibrium in vivo, with secretion and clearance of t-PA and PAI-1, and the inhibition reaction. Since this quilibrium is not the same in a plasma sample, the prior art assay may not determine the PAI-1 activity in vivo under physiological conditions. Thus, PAI-1 activity determined in a plasma sample can be lower than that in vivo, depending upon the original t-PA activity in vivo.
This prior art assay has the further drawback in that the values produced are highly dependent upon the standard curve and the accuracy of the t-PA activity used in the assays, requiring that new standard curves be generated with new samples.
Accordingly, an assay which can be employed to accurately measure PAI-1 and t-PA activity in vivo as well as in vitro, and the half-life of t-PA activity in a sample is desirable.
SUMMARY OF THE INVENTION
The invention relates to a method for assaying the PAI-1 activity and/or t-PA activity in a sample comprising measuring the activity of t-PA in the sample as a function of time and obtaining the PAI-1 and/or the t-PA activity from the second order rate equation, described below. The half-life of t-PA activity in an individual can also be determined from the assay. The invention includes methods of assaying or determining the PAI-1 and t-PA activities and the half-life of t-PA activity in a sample and kits therefor.
The invention provides an accurate assay for the determination of PAI-1 and t-PA activity in a sample which is not encumbered by the preparation of a new standard curve for t-PA activity with a new sample.
In one embodiment the invention includes a method for assaying the PAI-1 activity and/or t-PA activity in a sample wherein the sample is a body fluid comprising the steps of:
(a) adding tissue plasminogen activator to the sample;
(b) acidifying the sample;
(c) contacting a first preselected amount of the sample with plasminogen, thereby obtaining a mixture:
(d) maintaining the mixture under conditions sufficient to permit the t-PA-catalyzed conversion of plasminogen to plasmin;
(e) measuring the amount of plasmin formed, thereby measuring the activity of t-PA, (tPA) at a first time, t;
(f) repeating steps (c) through (d) at least at two different times, t
(g) employing the (tPA) at the first and second times, t, in the formula:
kt=−1/((PAI)
0
−(tPA)
0
)ln{(tPA)
0
[(PAI)
0
−(tPA)
0+(tPA)]}/(PAI)
0
(tPA)
wherein (PAI)
0
is the initial activity of PAI-1 at time zero;
(tPA)
0
is the initial activity of t-PA at time zero; and
(tPA) is the activity of t-PA at time t;
k is the second order rate constant for the reaction:
at assay conditions; and
(h) obtaining the values of (PAI-1)
0
and/or (tPA)
0
therefrom.
DETAILED DESCRIPTION OF THE INVENTION
The assay of the invention measures the activity of t-PA in a sample as a function of time and then calculates the activities of PAI-1 and/or t-PA at time zero, employing the second order reaction mechanism of PAI-1 and t-PA. The reaction is depicted by the formula.
Wherein k is the second order rate constant for the reaction. The second order rate constant can be calculated by equation I:
−d(t-PA)/dt=k(t-PA)(PAI)TM I
The second order rate constant (k) can be determined by plotting a series of pseudo-first-order rate constants of k (PAI-1) according to equation I. The second order rate constant will vary as a function of temperature, as is known in the art. For example, at room temperature, the second order rate constant, k, was determined by plotting a series of pseudo-first-order rate constant of k(PAI), approximately −0.014 U
−1
min
−1
under the conditions where PAI-1 is expressed in U/ml and t-PA activity is expressed as A405 (O.D.), as described below in the exemplification.
With this calculation, the PAI-1 activity at time zero and t-PA activity at time zero can be determined employing the above rate equation (II). The second order rate equation (II) for the reaction is:
kt=−1/((PAI)
0
−(tPA)
0
)ln{(tPA)
0
[(PAI)
0
−(tPA)
0
+(tPA)]}/(PAI)
0
(tPA)TM II
wherein t=time; (PAI)
0
is the initial activity of PAI-1 at time zero;
(tPA)
0
is the initial activity of t-PA at time zero; and
(tPA) is the activity of t-PA at time t.
PAI-1 activity is measured as U/ml (units per milliliter, in contrast to “arbitrary units” discussed above). 1 U/ml of PAI-1is the amount of PAI-1 required to inhibit 1 U/ml of t-PA. Generally 1 U/ml as determined herein, is the same or similar to 1 AU/ml. Upon determining the second order rate constant for the assay conditions to be employed, generation of a new standard curve for each assay can be avoided in the present assay.
Employing the assay, one can determine the activity of t-PA and/or PAI-1 at time zero from the rate equation (II). The assay can also be advantageously employed to measure the half-life of endogenous t-PA activity or exogenously added t-PA activity. In this embodiment, the time, t, where (t-PA) is ½ (t-PA)
0
, is calculated from the rate equation (II).
Samples which can be assayed for PAI-1 activity according to the claimed method include body fluids, such as blood, plasma, serum, urine or saliva. For example, freshly drawn blood plasma or serum can be assayed.
Where the sample to be assayed is blood, it is advantageous to centrifuge the sample, as is known in the art.
Preferably, the sample is acidified with a suitable buffer to achieve a pH between about 3.5 to about 4.5, most preferably about 4, with the appropriate ionic strength. An appropriate ionic strength can be between about 0.2 M to about 0.5 M, such as about 0.3 M. Acidification of the sample advantageously results in the inhibition of the t-PA-PAI-1 reaction.
Preferred buffers can include those which will not cause precipitation, such as protein precipitation, and will result in the acidification of the sample to the desired pH. An example of a suitable buffer is sodium acetate.
Optionally, t-PA is added to the sample. An example of where t-PA addition can be avoided is where the patient has, or is expected to have, high t-PA activity, such as, where the blood sample is drawn after venous occlusion. t-PA activity in these samples is generally high. In this case, the endogenous t-PA activity is measured as a function of time. t-PA can be added to the sample advantage
Lee Myoung H.
Mankowski Marcin J.
McDonagh Jan
Beth Israel Deaconess Medical Center Inc.
Gitomer Ralph
Hamilton Brook Smith & Reynolds P.C.
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